There are two related projects in this Program. Project 1. Single cell transcriptional profiling of primitive mouse hematopoietic cell populations. We have determined that lineage profiles are determined for megakaryocytic, lymphocytes, neutrophils and monocytes are specified in the earliest populations of hematopoietic progenitor cells while the erythroid lineage emerges from the megakaryocytic lineage at the Common myeloid progenitor stage. We are currently developing methods to prospectively isolate these populations for more in-depth study. Project 2. Defining the transcriptional and epigenetic profiles of differentiating mouse hematopoietic cells. We will determine the chromatin accessibility as well as the methylation and acetylation of histone proteins across the genome and correlated these with mRNA expression and DNA methylation. In mammals, the number of HSC per animal is remarkably similar between species despite great differences in the number of cells produced. Hematopoiesis has traditionally been modeled as a hierarchy in which the progeny of HSC become progressively committed to a branch (myeloid or lymphoid) and ultimately to a single lineage in a stepwise fashion. Recently single cell analyses and improved colony assays have shown that hematopoiesis follows a more fluid process where lineages are specified earlier in hematopoiesis than the original hierarchical model predicted. For example, we found that the human MEP population contains three subpopulations of lineage primed cells that could be prospectively separated by surface marker expression. Project 1 is to perform single cell transcriptional profiling of primitive mouse hematopoietic cell populations to determine the stage of hematopoiesis where commitment to the erythroid and other lineages becomes specified. The identification of primitive, lineage committed cells will allow us to ask when the epigenetic marks present in mature hematopoietic cells first appear. We have completed a single cell profile of over 10,00 cells each from primitive hematopoietic stem and progenitor cells (LSK), Common Myeloid Progenitors (CMP), Megakaryocyte/Erythroid progenitors (MEP) and Granulocyte/Monocyte progenitors (GMP). In these populations we have identified 25 distinct transcriptional profiles that segregate with the stage (e.g. CMP or GMP) as opposed to lineage. We have shown that the erythroid lineage is the last to emerge, following the lymphoid, granulocytic and monocyte and lymphoid lineages. We are currently assigning a surface marker profile to each population for prospective isolation. Project 2 is to define the transcriptional and epigenetic profiles of differentiating mouse hematopoietic cells and to test whether these patterns are established in more primitive cells. This ambitious, hypothesis generating goal is well beyond the capacities of any single lab and we have joined forces with the ValIdated Systematic IntegratiON of hematopoietic epigenomes consortium (VISION) which is a group of 10 different laboratories with the necessary wide range of expertise. Our involvement with VISION began in 2010 when we defined epigenetic changes associated with erythropoiesis in primary mouse hematopoietic cells. In the next four years we plan to expand our analyses to include non-erythroid hematopoietic cell types to better define the specific changes associated with the differentiation of erythroid cells. Our group (Drs. Jens Lichtenberg and Elisabeth Heuston) will develop transcriptional, DNA methylation and histone modification profiles that complement data generated in the laboratories of Mitchell Weiss (St. Jude Childrens Research Hospital), Gerd Blobel (University of Pennsylvania), Jim Hughes (Oxford University) and Doug Higgs (Oxford University) to define the chromatin landscape during hematopoietic differentiation. These data will be integrated into a comprehensive 3D profile by the computational arm of VISION, which includes Jens Lichtenberg from our group, Berthold Gottgens (University of Cambridge), James Taylor (Johns Hopkins), Feng Yue (Penn State Hershey), Yu Zhang (Penn State) and Ross Hardison (Penn State). We have shown that the epigenetic profile of megakaryocytic is established in the most primitive hematopoietic stem and progenitor cells and is maintained during differentiation. In contrast, the epigenetic profile of erythroblasts is acquired during differentiation. The rationale behind our choice of the mouse system was carefully considered. Our studies are intended to complement work being performed by ENCODE to define the epigenetic landscape in human hematopoietic cells. Mouse and human hematopoiesis, while highly conserved in some respects, also differ in many significant ways. By comparing the mouse and human epigenetic profiles, we will identify overlapping (common) patterns as well as distinct patterns that can be associated with the different properties of mouse and human erythropoiesis, which will generate more informed hypotheses than would be possible by studying hematopoiesis in a single species. For example, haploinsufficiency of ribosomal proteins in humans leads to a block in erythropoiesis resulting in Diamond Blackfan anemia, while haploinsufficiency of ribosomal proteins in mice is benign. Identifying the differences in mouse and human RP gene regulation may identify a mouse like pathway that could be targeted to treat DBA patients.